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Assessment of pan-Leishmania detection by recombinase polymerase amplification assay

Louizi C, Khan MAA, Faisal K, Chowdhury R, Ghosh P, Hossain F, et al.

Diagn Microbiol Infect Dis, 2023 Feb;105(2):115862.
PMID: 36493571 DOI: 10.1016/j.diagmicrobio.2022.115862

The spread of vector habitats along with increasing human mobility can introduce atypical Leishmania species and hence can challenge existing diagnostic practices for rapid detection of active infection with species outside the narrow target range. Here we assessed the pan-Leishmania detection ability of isothermal recombinase polymerase amplification (RPA) assays targeting 18S rRNA gene, cathepsin L-like cysteine proteinase B (Cpb) gene, and kinetoplast minicircle DNA (kDNA) regions. While the lowest limit of detection of the 18S rRNA-RPA and Cpb-RPA assays were estimated as 12 and 17 standard DNA molecules, respectively, both assays could amplify genomic DNA of 7 pathogenic Leishmania species. Evaluation of 18S rRNA-RPA and our previously developed kDNA-RPA assays on 70 real-time PCR-positive leishmaniasis samples of varying pathologies resulted in sensitivity rates of 35.71% and 88.57%, respectively, while the combined sensitivity was 98.57%. Combinatorial application of 18S rRNA-RPA and kDNA-RPA assays can be recommended for further diagnostic assessments.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods
Telomerase activity in Malaysian patients with central nervous system tumors

Isa MN, Sulong S, Sidek MR, George PJ, Abdullah JM

Southeast Asian J. Trop. Med. Public Health, 2003 Dec;34(4):872-6.
PMID: 15115103

Telomerase, the enzyme that stabilizes telomere length is reactivated with almost all cancer types, and may be a useful diagnostic marker for malignancy. Telomerase activity has been detected in germ line cells and most cancer cells, whereas most normal somatic cells have no clearly detectable telomerase activity. In our study, we aim to detect telomerase activity in 20 human central nervous system tumors from Malaysian patients. Telomerase activity was detected based on a highly sensitive procedure consisting of a CHAPS detergent-based extraction from frozen tissues and a PCR-based telomeric repeat amplification protocol (TRAP) using a TRAPEZE Telomerase Detection Kit (Intergen, Co). Telomerase activity was considered positive when a ladder of products was observed starting at 50bp, with 6bp increments. The activity was detected in 30% of the samples analysed, included glioblastoma multiforme, meduloblastoma, paraganglioma and oligodendroglioma. The result of Fisher's exact test indicated that there was a significant association between telomerase activity status with tumor grade (p=0.003). These results suggest that telomerase activity may be an important marker for tumor malignancy.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods
Fulltext Detection of dengue viruses using reverse transcription-loop-mediated isothermal amplification

Teoh BT, Sam SS, Tan KK, Johari J, Danlami MB, Hooi PS, et al.

BMC Infect Dis, 2013;13:387.
PMID: 23964963 DOI: 10.1186/1471-2334-13-387

BACKGROUND: Early and rapid detection of dengue virus (DENV) infection during the febrile period is crucial for proper patient management and prevention of disease spread. An easy to perform and highly sensitive method is needed for routine implementation especially in the resource-limited rural healthcare settings where dengue is endemic.
METHODS: A single-tube reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay with a set of nine primers was developed for the detection of all four DENV serotypes and their different genotypes. The sensitivity and specificity of the RT-LAMP were evaluated. The clinical applicability of RT-LAMP assay for detection of DENV RNA was assessed in a total of 305 sera of clinically-suspected dengue patients. The test results of RT-LAMP were statistically compared to those of quantitative reverse transcription-polymerase chain reaction (qRT-PCR), IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA).
RESULTS: Acute DENV infection was confirmed in 171 samples (n = 305); 43.3% (74/171) and 46.8% (80/171) of the samples were positive for DENV using RT-LAMP and qRT-PCR, respectively. The combination of RT-LAMP with the dengue IgM and IgG ELISA increased detection of acute DENV infection to 97.7% (167/171), in comparison to only 70.8% (121/171) when dengue IgM and IgG ELISA alone were used. The RT-LAMP assays showed high concordance (κ = 0.939) with the qRT-PCR. The RT-LAMP assay detected up to 10 copies of virus RNA within an hour but 100% reproducibility (12/12) was achieved with 100 copies. There was no cross reactivity of RT-LAMP with other closely related arboviruses.
CONCLUSION: The RT-LAMP assay developed in this study is sensitive, specific and simple to perform. The assay improved the detection of dengue when used in combination with serological methods.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
Fulltext Colorimetric Detection of Dengue by Single Tube Reverse-Transcription-Loop-Mediated Isothermal Amplification

Lau YL, Lai MY, Teoh BT, Abd-Jamil J, Johari J, Sam SS, et al.

PLoS One, 2015;10(9):e0138694.
PMID: 26384248 DOI: 10.1371/journal.pone.0138694

Dengue is usually diagnosed by isolation of the virus, serology or molecular diagnostic methods. Several commercial kits for the diagnosis of dengue are existing, but concerns have arisen regarding to the affordability and performance characteristics of these kits. Hence, the loop-mediated isothermal amplification (LAMP) is potentially ideal to be used especially in resource limited environments. Serum was collected from healthy donors and patients diagnosed with dengue infection. RNA extracted from the serum samples were tested by reverse-transcription-LAMP assay developed based on 3'-NCR gene sequences for DENV 1-4. Results were interpreted by a turbidity meter in real time or visually at the end of the assay. Sensitivity and specificity of RT-LAMP results were calculated and compared to qRT-PCR and ELISA. RT-LAMP is highly sensitive with the detection limit of 10 RNA copies for all serotypes. Dengue virus RNA was detected in all positive samples using RT-LAMP and none of the negative samples within 30-45 minutes. With continuing efforts in the optimization of this assay, RT-LAMP may provide a simple and reliable test for detecting DENV in areas where dengue is prevalent.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
Fulltext A reverse transcription loop-mediated isothermal amplification for broad coverage detection of Asian and African Zika virus lineages

Teoh BT, Chin KL, Samsudin NI, Loong SK, Sam SS, Tan KK, et al.

BMC Infect Dis, 2020 Dec 11;20(1):947.
PMID: 33308203 DOI: 10.1186/s12879-020-05585-4

BACKGROUND: Early detection of Zika virus (ZIKV) infection during the viremia and viruria facilitates proper patient management and mosquito control measurement to prevent disease spread. Therefore, a cost-effective nucleic acid detection method for the diagnosis of ZIKV infection, especially in resource-deficient settings, is highly required.
METHODS: In the present study, a single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of both the Asian and African-lineage ZIKV. The detection limit, strain coverage and cross-reactivity of the ZIKV RT-LAMP assay was evaluated. The sensitivity and specificity of the RT-LAMP were also evaluated using a total of 24 simulated clinical samples. The ZIKV quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used as the reference assay.
RESULTS: The detection limit of the RT-LAMP assay was 3.73 ZIKV RNA copies (probit analysis, P ≤ 0.05). The RT-LAMP assay detected the ZIKV genomes of both the Asian and African lineages without cross-reacting with other arthropod-borne viruses. The sensitivity and specificity of the RT-LAMP assay were 90% (95% CI = 59.6-98.2) and 100% (95% CI = 78.5-100.0), respectively. The RT-LAMP assay detected ZIKV genome in 9 of 24 (37.5%) of the simulated clinical samples compared to 10 of 24 (41.7%) by qRT-PCR assay with a high level of concordance (κ = 0.913, P

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
The reliability of a rapid molecular detection method in determining the prevalence of rifampicin-resistant Mycobacterium tuberculosis in an urban district health facility in Malaysia

Ding CH, Ismail Z, Sulong A, Wahab AA, Gan B, Mustakim S, et al.

Malays J Pathol, 2020 Dec;42(3):401-407.
PMID: 33361721

INTRODUCTION: Rifampicin is a key first-line antimycobacterial agent employed for the treatment of pulmonary tuberculosis (PTB). This study sought to obtain prevalence data on rifampicin-resistant Mycobacterium tuberculosis among smear-positive PTB patients in the Klang District of Malaysia.
MATERIALS AND METHODS: A total of 103 patients from the Chest Clinic of Hospital Tengku Ampuan Rahimah with sputum smears positive for acid-fast bacilli were included in this cross-sectional study. All sputa were tested using Xpert MTB/RIF to confirm the presence of M. tuberculosis complex and detect rifampicin resistance. Sputa were also sent to a respiratory medicine institute for mycobacterial culture. Positive cultures were then submitted to a reference laboratory, where isolates identified as M. tuberculosis complex underwent drug susceptibility testing (DST).
RESULTS: A total of 58 (56.3%) patients were newly diagnosed and 45 (43.7%) patients were previously treated. Xpert MTB/RIF was able to detect rifampicin resistance with a sensitivity and specificity of 87.5% and 98.9%, respectively. Assuming that a single resistant result from Xpert MTB/RIF or any DST method was sufficient to denote resistance, a total of 8/103 patients had rifampicinresistant M. tuberculosis. All eight patients were previously treated for PTB (p<0.05). The overall prevalence of rifampicin resistance among smear-positive PTB patients was 7.8%, although it was 17.8% among the previously treated ones.
CONCLUSION: The local prevalence of rifampicin-resistant M. tuberculosis was particularly high among previously treated patients. Xpert MTB/RIF can be employed in urban district health facilities not only to diagnose PTB in smear-positive patients, but also to detect rifampicin resistance with good sensitivity and specificity.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
Fulltext Prenatal diagnosis of aneuploidies in amniotic fluid by multiple ligation-dependent probe amplification (MLPA) analysis

Hamidah NH, Munirah AR, Hafiza A, Farisah AR, Shuhaila A, Norzilawati MN, et al.

Malays J Pathol, 2014 Dec;36(3):163-8.
PMID: 25500514 MyJurnal

Prenatal diagnosis is essential in the new era of diagnosis and management of genetic diseases in obstetrics. Multiple ligation-dependent probe amplification (MLPA) is a recent technique for prenatal diagnosis for the relative quantification of 40 different nucleic acid sequences in one single reaction. We had utilized the MLPA technique in detecting aneuploidies in amniotic fluid samples from 25 pregnant women from the Obstetrics and Gynaecology Department UKMMC, versus the quantitative fluorescent polymerase chain reaction (QF-PCR) method. Conclusive results were obtained in 18 cases and all were concordant with that of the QF-PCR. All four cases of trisomies were correctly identified including one case with maternal cell contamination.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
Fulltext A family study of HbS in a Malay family by molecular analysis

Hafiza A, Noor HH, Noor FA, Azlin I, Ainoon O

Malays J Pathol, 2010 Dec;32(2):137-41.
PMID: 21329186 MyJurnal

Sickle cell disease (SCD) is an inherited red cell disorder, characterized by the tendency of haemoglobin S or sickle haemoglobin to polymerize and assume a characteristic sickle shape. Molecular analysis has been the mainstay of detection method when confirmation is required. Previously a polymerase chain reaction (PCR)-based restriction enzyme analysis was used for this purpose. A simple bidirectional allele-specific amplification, recently described by Waterfall in 2001 was used to detect the GAG --> GTG mutation on codon 6 of the beta globin gene. Two sets of primers for the mutant and the wild type alleles were used in a single PCR reaction to amplify the regions of interest. The resultant PCR products will produce two fragments at 517 and 267 base pair (bp) respectively. This report highlights the investigations for SCD in the family of a 16-year old girl with recurrent painful crisis affecting the lower limbs whereby the family members are asymptomatic for the disease. Her haemoglobin electrophoresis at an alkaline pH showed dense bands at the HbS and HbF regions, while her father and two sisters had bands at HbS, HbF and HbA. The PCR analysis showed that she was homozygous for the mutation by the presence of only one band at 267 bp fragment, while the father and her sisters were heterozygotes, with the presence of two bands at 267 as well as 517 bp fragments. DNA sequencing of the sample confirmed the mutation. In conclusion, this case report highlighted the simple and cheap yet practical method for molecular confirmation of the presence of HbS gene in subjects with homozygous or heterozygous state of the condition.

Matched MeSH terms: Nucleic Acid Amplification Techniques
Fulltext Optimization of Polymerase Chain Reaction (PCR) of mitochondrial cytochrome c oxidase I (COI) gene in two Bornean fanged frogs

Ramlah Zainudin, Augustine Gawin, Dency Flenny

Pertanika Journal of Science & Technology, 2011;19(1):57-66.
MyJurnal

Limnonectes kuhlii and Limnonectes leporinus are two of the Bornean fanged frogs (without advertisement call) which are widely distributed, thus thought to exhibit different evolutionary lineages and the existence of genetically cryptic species. Yet, the two species are still under study especially at the molecular level. Hence, cytochrome c oxidase I (COI) of mitochondrial gene was used to investigate suitable parameters for DNA amplification using the Polymerase Chain Reaction (PCR) method. Three PCR programmes (varied in the temperatures and period of each PCR step) were employed to identify the most efficient parameters in amplifying PCR products for both species. From the three programmes, Programme B (Initial denaturation: 96°C for 5 min; denaturation: 95°C for 45 sec; annealing: 48-53°C for 1 min 30 sec; extension: 72°C for 1 min 30 sec; final extension: 72°C for 10 min, 30 cycles) showed the highest percentage (53%) of optimal PCR products. The other two programmes showed non-specific products or “primer-dimers”. The results also suggest that the annealing temperature of 52°C, 0.025-0.05 units/µl of 1.5mM Taq polymerase, 0.04 mM of
dNTPs mix and optimal concentrations of magnesium in 50 µl of reaction mixture were sufficient enough to amplify high quality PCR products for both species. However, using Programme B, the re-amplification of the PCR products yielded “primer-dimer”. In addition, a ‘Hot-Start’ PCR method was also applied and mostly yielded in an optimal PCR amplification. Nevertheless, further research on the second amplification of the two species should be conducted to determine the causes of the primer-dimer production.

Matched MeSH terms: Nucleic Acid Amplification Techniques
DNA/Nano based advanced genetic detection tools for authentication of species: Strategies, prospects and limitations

Khalil I, Hashem A, Nath AR, Muhd Julkapli N, Yehye WA, Basirun WJ

Mol Cell Probes, 2021 10;59:101758.
PMID: 34252563 DOI: 10.1016/j.mcp.2021.101758

Authentication, detection and quantification of ingredients, and adulterants in food, meat, and meat products are of high importance these days. The conventional techniques for the detection of meat species based on lipid, protein and DNA biomarkers are facing challenges due to the poor selectivity, sensitivity and unsuitability for processed food products or complex food matrices. On the other hand, DNA based molecular techniques and nanoparticle based DNA biosensing strategies are gathering huge attention from the scientific communities, researchers and are considered as one of the best alternatives to the conventional strategies. Though nucleic acid based molecular techniques such as PCR and DNA sequencing are getting greater successes in species detection, they are still facing problems from its point-of-care applications. In this context, nanoparticle based DNA biosensors have gathered successes in some extent but not to a satisfactory stage to mark with. In recent years, many articles have been published in the area of progressive nucleic acid-based technologies, however there are very few review articles on DNA nanobiosensors in food science and technology. In this review, we present the fundamentals of DNA based molecular techniques such as PCR, DNA sequencing and their applications in food science. Moreover, the in-depth discussions of different DNA biosensing strategies or more specifically electrochemical and optical DNA nanobiosensors are presented. In addition, the significance of DNA nanobiosensors over other advanced detection technologies is discussed, focusing on the deficiencies, advantages as well as current challenges to ameliorate with the direction for future development.

Matched MeSH terms: Nucleic Acid Amplification Techniques
Rapid in-situ detection kit (RisK): Development of loop-mediated isothermal amplification (LAMP) assay for the rapid identification of selected invasive alien fish in Malaysian freshwaters

Vythalingam LM, Hossain MAM, Bhassu S

Mol Cell Probes, 2021 02;55:101683.
PMID: 33259896 DOI: 10.1016/j.mcp.2020.101683

Invasive alien fish species have become a silent treat towards the ecosystem especially the native fish population in Malaysia. There has been a need to develop rapid identification methods that can aid management teams in identifying fish species that are not native to our ecosystem. Current visual identification methods are highly tedious and require time, delaying action towards curbing the invasion. The LAMP assay successfully identified six popular invasive fish species in Malaysia. None of the LAMP assays showed false positives and the Limit of Detection of the LAMP primers were highly sensitive and could detect DNA samples up to 1 × 10-15 ng/μl. The LAMP primers designed were highly specific to the target species and did not amplify non target species. DNA sequencing was done to ensure the accuracy of LAMP assay results. This study demonstrates that LAMP is a suitable tool in species identification efforts of invasive fish species in Malaysia.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
Streptococcus gallolyticus infection: A neglected marker for colorectal cancer?

Bin Ismail CMKH, Bin Mohammad Aidid E, Binti Hamzah HA, Bin Shalihin MSE, Bin Md Nor A

Arab J Gastroenterol, 2023 Aug;24(3):163-167.
PMID: 37156704 DOI: 10.1016/j.ajg.2023.02.002

BACKGROUND AND STUDY AIMS: Colorectal cancer (CRC) is the second most common cancer in Malaysia and mostly detected at advanced stages due to lack of awareness of CRC symptoms and signs. CRC pathogenesis is multifactorial, and there is ambiguous evidence on association of Streptococcus gallolyticus infection with CRC that needs further attention. Thus, a case-control study was conducted to determine whether S. gallolyticus infection is a predictor for CRC occurrence among patients attending Sultan Ahmad Shah Medical Centre@IIUM (SASMEC@IIUM).
PATIENTS AND METHODS: A total of 33 stool samples from patients diagnosed with CRC and 80 from patients without CRC attending surgical clinic of SASMEC@IIUM were collected and analyzed with iFOBT test and PCR assay to detect S. gallolyticus.
RESULTS: In this study, the proportion of S. gallolyticus infection was higher among patients with CRC (48.5%) compared with the control group (20%). Univariate analysis shows that occult blood in stool, S. gallolyticus infection and family history were significantly associated with the development of CRC (P

Matched MeSH terms: Nucleic Acid Amplification Techniques
Fulltext Real-time PCR evaluation of seven DNA extraction methods for the purpose of GMO analysis

Jasbeer, K., Son, R., Mohamad Ghazali, F., Cheah, Y.K.

International Food Research Journal, 2009;16(3):329-341.
MyJurnal

Successful DNA amplification is vital for the detection of specific DNA targets in feeds, and this in return depends on the ability of DNA extraction methods to produce good quality DNA. In this study, seven methods were compared for DNA extraction from feeds using quantitative polymerase chain reaction (PCR) of single copy maize (Zea mays) endogenous hmg (high mobility group) gene. Relative levels of hmg were used to evaluate the DNA quality. Spectrophotometer determination of DNA was also carried out to assess DNA yield and DNA purity, while electrophoretic analysis of genomic DNA extracts was carried out to investigate DNA integrity. The findings illustrate that the DNA extraction methods have a significant effect on DNA quality. Statistically, the Epicentre method extracted the highest DNA yield while the Wizard method had the lowest DNA yield with high DNA purity and integrity. However, the Wizard method recovered the most amplifiable DNA per reaction, indicating that template quality and integrity had greater influence over hmg amplification than DNA yield.

Matched MeSH terms: Nucleic Acid Amplification Techniques
Fulltext Diagnostic accuracy of genetic markers and nucleic acid techniques for the detection of Leptospira in clinical samples: A meta-analysis

Lam JY, Low GK, Chee HY

PLoS Negl Trop Dis, 2020 02;14(2):e0008074.
PMID: 32049960 DOI: 10.1371/journal.pntd.0008074

BACKGROUND: Leptospirosis is often difficult to diagnose because of its nonspecific symptoms. The drawbacks of direct isolation and serological tests have led to the increased development of nucleic acid-based assays, which are more rapid and accurate. A meta-analysis was performed to evaluate the diagnostic accuracy of genetic markers for the detection of Leptospira in clinical samples.
METHODOLOGY AND PRINCIPLE FINDINGS: A literature search was performed in Scopus, PubMed, MEDLINE and non-indexed citations (via Ovid) by using suitable keyword combinations. Studies evaluating the performance of nucleic acid assays targeting leptospire genes in human or animal clinical samples against a reference test were included. Of the 1645 articles identified, 42 eligible studies involving 7414 samples were included in the analysis. The diagnostic performance of nucleic acid assays targeting the rrs, lipL32, secY and flaB genes was pooled and analyzed. Among the genetic markers analyzed, the secY gene showed the highest diagnostic accuracy measures, with a pooled sensitivity of 0.56 (95% CI: 0.50-0.63), a specificity of 0.98 (95% CI: 0.97-0.98), a diagnostic odds ratio of 46.16 (95% CI: 6.20-343.49), and an area under the curve of summary receiver operating characteristics curves of 0.94. Nevertheless, a high degree of heterogeneity was observed in this meta-analysis. Therefore, the present findings here should be interpreted with caution.
CONCLUSION: The diagnostic accuracies of the studies examined for each genetic marker showed a significant heterogeneity. The secY gene exhibited higher diagnostic accuracy measures compared with other genetic markers, such as lipL32, flaB, and rrs, but the difference was not significant. Thus, these genetic markers had no significant difference in diagnostic accuracy for leptospirosis. Further research into these genetic markers is warranted.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
Fulltext Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

Wong YP, Othman S, Lau YL, Radu S, Chee HY

J Appl Microbiol, 2018 Mar;124(3):626-643.
PMID: 29165905 DOI: 10.1111/jam.13647

Loop-mediated isothermal amplification (LAMP) amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions by using a DNA polymerase with high displacement strand activity and a set of specifically designed primers to amplify targeted DNA strands. Following its first discovery by Notomi et al. ( Nucleic Acids Res 28: E63), LAMP was further developed over the years which involved the combination of this technique with other molecular approaches, such as reverse transcription and multiplex amplification for the detection of infectious diseases caused by micro-organisms in humans, livestock and plants. In this review, available types of LAMP techniques will be discussed together with their applications in detection of various micro-organisms. Up to date, there are varieties of LAMP detection methods available including colorimetric and fluorescent detection, real-time monitoring using turbidity metre and detection using lateral flow device which will also be highlighted in this review. Apart from that, commercialization of LAMP technique had also been reported such as lyophilized form of LAMP reagents kit and LAMP primer sets for detection of pathogenic micro-organisms. On top of that, advantages and limitations of this molecular detection method are also described together with its future potential as a diagnostic method for infectious disease.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
Fulltext Specific, sensitive and rapid detection of human plasmodium knowlesi infection by loop-mediated isothermal amplification (LAMP) in blood samples

Lau YL, Fong MY, Mahmud R, Chang PY, Palaeya V, Cheong FW, et al.

Malar J, 2011;10:197.
PMID: 21774805 DOI: 10.1186/1475-2875-10-197

The emergence of Plasmodium knowlesi in humans, which is in many cases misdiagnosed by microscopy as Plasmodium malariae due to the morphological similarity has contributed to the needs of detection and differentiation of malaria parasites. At present, nested PCR targeted on Plasmodium ssrRNA genes has been described as the most sensitive and specific method for Plasmodium detection. However, this method is costly and requires trained personnel for its implementation. Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method was developed for the clinical detection of P. knowlesi. The sensitivity and specificity of LAMP was evaluated in comparison to the results obtained via microscopic examination and nested PCR.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
Fulltext Specific, sensitive, and rapid diagnosis of active toxoplasmosis by a loop-mediated isothermal amplification method using blood samples from patients

Lau YL, Meganathan P, Sonaimuthu P, Thiruvengadam G, Nissapatorn V, Chen Y

J Clin Microbiol, 2010 Oct;48(10):3698-702.
PMID: 20660217 DOI: 10.1128/JCM.00462-10

Loop-mediated isothermal amplification (LAMP), a rapid nucleic acid amplification method, was developed for the clinical diagnosis of toxoplasmosis. Three LAMP assays based on the SAG1, SAG2, and B1 genes of Toxoplasma gondii were developed. The sensitivities and specificities of the LAMP assays were evaluated by comparison with the results of conventional nested PCR. The LAMP assays were highly sensitive and had a detection limit of 0.1 tachyzoite, and no cross-reactivity with the DNA of other parasites was observed. Blood was collected from 105 individuals to test the LAMP assays: 40 patients with active toxoplasmosis, 40 negative controls, and 25 patients with other parasitic infections. The SAG2-based LAMP (SAG2-LAMP) had a greater sensitivity (87.5%) than the SAG1-LAMP (80%), B1-LAMP (80%), and nested PCR (62.5%). All the LAMP assays and nested PCR were 100% specific. This is the first report of a study which applied the LAMP method to diagnose toxoplasmosis from human blood samples. Due to its simplicity, sensitivity, and specificity, LAMP is suggested as an appropriate method for routine diagnosis of active toxoplasmosis in humans.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
Evaluation of serological transfusion-transmitted viral diseases and mutliplex nucleic acid testing in malaysian blood donors

Yaseen SG, Ahmed SA, Johan MF, Kiron R, Daher AM

Transfus Apher Sci, 2013 Dec;49(3):647-51.
PMID: 23890575 DOI: 10.1016/j.transci.2013.07.003

Transmission of infectious diseases is a recognized complication of blood transfusion and blood products. Nucleic acid testing (NAT) may contribute to improved efficiency of blood screening and thereby increase the safety margin for transfused blood.

Matched MeSH terms: Nucleic Acid Amplification Techniques/instrumentation; Nucleic Acid Amplification Techniques/methods*
Isothermal amplifications - a comprehensive review on current methods

Glökler J, Lim TS, Ida J, Frohme M

Crit Rev Biochem Mol Biol, 2021 12;56(6):543-586.
PMID: 34263688 DOI: 10.1080/10409238.2021.1937927

The introduction of nucleic acid amplification techniques has revolutionized the field of medical diagnostics in the last decade. The advent of PCR catalyzed the increasing application of DNA, not just for molecular cloning but also for molecular based diagnostics. Since the introduction of PCR, a deeper understanding of molecular mechanisms and enzymes involved in DNA/RNA replication has spurred the development of novel methods devoid of temperature cycling. Isothermal amplification methods have since been introduced utilizing different mechanisms, enzymes, and conditions. The ease with which isothermal amplification methods have allowed nucleic acid amplification to be carried out has had a profound impact on the way molecular diagnostics are being designed after the turn of the millennium. With all the advantages isothermal amplification brings, the issues or complications surrounding each method are heterogeneous making it difficult to identify the best approach for an end-user. This review pays special attention to the various isothermal amplification methods by classifying them based on the mechanistic characteristics which include reaction formats, amplification information, promoter, strand break, and refolding mechanisms. We would also compare the efficiencies and usefulness of each method while highlighting the potential applications and detection methods involved. This review will serve as an overall outlook on the journey and development of isothermal amplification methods as a whole.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods*
Fulltext A Sensitive Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Direct Visual Detection of SARS-CoV-2

Lau YL, Ismail IB, Izati Binti Mustapa N, Lai MY, Tuan Soh TS, Hassan AH, et al.

Am J Trop Med Hyg, 2020 Dec;103(6):2350-2352.
PMID: 33098286 DOI: 10.4269/ajtmh.20-1079

A simple and rapid reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of SARS-CoV-2. The RT-LAMP assay was highly specific for SARS-CoV-2 and was able to detect one copy of transcribed SARS-CoV-2 RNA within 24 minutes. Assay validation performed using 50 positive and 32 negative clinical samples showed 100% sensitivity and specificity. The RT-LAMP would be valuable for clinical diagnosis and epidemiological surveillance of SARS-CoV-2 infection in resource-limited areas as it does not require the use of sophisticated and costly equipment.

Matched MeSH terms: Nucleic Acid Amplification Techniques/methods; Nucleic Acid Amplification Techniques/standards*

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